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Aux/IAA interaction with BTB/POZ-MATH ubiquitin-ligases is conserved between Arabidopsis and Physcomitrella


Auxin is a phytohormone known for its diverse control in regulating growth, development, and environmental responses. Auxin signaling occurs through transcriptional regulation facilitated by auxin-response transcription factors (ARFs), ARF-repressing Auxin/INDOLE-3-ACETIC ACID (Aux/IAA) proteins, and a ubiquitin-proteasome system (UPS) mediated by the SKP1-CULLIN1-F-Box (SCF) complex. SCF binds to Aux/IAAs in an auxin-dependent manner, targeting the Aux/IAAs for 26S proteasomal degradation. In the absence of Aux/IAAs ARFs are no longer repressed, allowing them to facilitate expression of downstream auxin-response genes. While Aux/IAAs are essential to auxin-mediated transcriptional regulation, the functions of many of the 29 Arabidopsis Aux/IAA family members remain unknown and how other ubiquitin-ligase systems may play a role is unclear. A yeast 2-hybrid (Y2H) library screen using Aux/IAA10 as bait identified the ligase BTB/POZ-MATH3 (BPM3) as a potential binding partner. Interestingly, BPM3 is known to assemble in a ubiquitin-ligase complex: the Cullin3 (CUL3)-based E3 complex (CRL3). In this thesis, I investigated whether CRL3BPM functions as a regulator of the Aux/IAAs. I assayed the physical interactions between BPM4 and the Aux/IAA family members via Y2H, confirming interaction with Aux/IAAs 6, 10, and 11 (IAA6/10/11). I demonstrated interaction from gene family members across divergent clades, which indicates that the BPM-Aux/IAA interaction is evolutionarily conserved. I confirmed interaction of BPM orthologs from the moss species Physcomitrella patens with all 3 Physcomitrella IAA orthologs, as well as with Arabidopsis IAA6/10/11 in subsequent Y2H assays. These results suggest that the BPM-Aux/IAA interaction is ancient, predating the bryophyte-tracheophyte divide. To address the biological significance of the BPM-Aux/IAA interaction, Arabidopsis double knock-out mutants aux/iaa10/11 were generated and I analyzed the auxin-induced hypocotyl elongation phenotype, finding no significant difference compared to wild-type plants. This result indicates that disruption of Aux/IAA10/11 expression is not sufficient to destabilize this specific phenotype.

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